Observational study on passive leg raising and the autonomic nervous system

Abstract In the intensive care and perioperative setting, circulation is often supported by intravenous fluid preceded by prediction of fluid responsiveness during a passive leg raising (PLR) maneuver. An increase in stroke volume (SV) or cardiac output (CO) of 10%–15% indicates that the subject may increase the flow upon volume expansion. However, the semi‐recumbent position as an initial position in PLR likely reduces SV by gravitational displacement of central blood volume (CBV) to lower extremities, thereby accentuating volume responsiveness during leg raising in healthy people. Coincident with gravitational perturbations in hemodynamics, remedial changes occur in the autonomic nervous system (ANS), as expressed in spectral power in heart rate variability (HRV). This study aims to clarify these concomitant changes during PLR. A convenience number of healthy volunteers (N = 11) were recruited by advertisement in university departments. The subjects were exposed to the established PLR sequence and the heart rate (HR), mean arterial pressure (MAP), SV, and CO were sampled at 1 Hz, while electrocardiogram was recorded at 1000 Hz. Relative powers reflecting autonomic nervous system activity were assessed from spectral analysis of HRV. In response to PLR, SV increased (12.4% ± 8.7%, p < 0.0026), while HR (−7.6% ± 4.7%, p < 0.0009) and MAP (−7.6% ± 6.9%, p < 0.01) decreased, with no change in CO (4.1% ± 12.8%, ns). The HRV low‐frequency component was reduced (−34%; p < 0.0095), while the high‐frequency activity increased (78.5%; p < 0.0013), with a 63% decrease in the low/high frequency ratio (p < 0.0078). Thus, HRV indicated a reduced sympathetic index (semi‐recumbent 0.808 vs. PLR −0.177 a.u., p < 0.001) and an increased parasympathetic index (−0.141 to 0.996 a.u., p < 0.0001). Gravitational depletion and expansion of CBV during PLR were associated with a counterregulatory autonomic response. Healthy volunteers appeared volume responsive in terms of SV, but not CO. Responses to PLR are influenced by the ANS, and HRV analysis should be included in the assessment of the PLR test.


| BACKGROUND
Management of the cardiovascular system in perioperative and intensive care has moved from "one size fits all" in terms of pressure, flow, and fluids to an individualized goal-directed approach based on-ideally-matching the oxygen delivery (DO 2 ) to the oxygen consumption (VO 2 ) using fluids, vasoactive, and/or inotropic therapy. To assess the option of volume expansion, an international peer assembly recently emphasized the use of dynamic indices in preload responsiveness, supplanting the indiscriminate use of a fluid bolus which at best elicits a positive response in 50% of patients (Michard & Teboul, 2002). Therefore, pulse pressure variation (PPV), stroke volume variation (SVV), passive leg raising (PLR), and end-expiratory occlusion test (EEO) are highly recommended as these measures can predict preload responsiveness (De Backer et al., 2019). PLR further carries the endorsement of using an internal fluid bolus avoiding the potentially deleterious effect of external fluid and has been promoted as "five rules and not a drop of fluid" referring to the positioning sequence and the mobilization of blood from the splanchnic area and lower extremities during the procedure (Monnet & Teboul, 2015). However, serious concerns have been raised regarding the physiological groundwork behind the use and interpretation of PPV and SVV (Aneman & Sondergaard, 2016;Bahlmann et al., 2016;Sondergaard, 2013). Likewise, the PLR maneuver deserves scrutiny to clarify its position in the ICU and the perioperative setting. HRV represented by the changes in relative proportions of spectral frequencies in relation to physiological changes (e.g., postural maneuvers) is well recognized and an established tool in research involving ANS (Malik et al., 2019). In this hypothesis-generating observational pilot study, the hemodynamic and ANS responses to PLR were examined in healthy subjects. The study was pragmatic, used commonly available monitoring equipment, digital signal analysis, and is intended as a precursor to studies in patients with attenuated ANS due to disease, anesthesia, and/or sedation. Sophisticated physiological examinations (e.g., lower body negative pressure, microneurography, electrical impedance tomography, and transcardiac/pulmonary thermodilution) were avoided to obtain general clinical applicability.

| Objective
This study aimed to register, describe, and interpret concomitant changes in hemodynamic variables and HRV during PLR.

| Test subjects
Eleven healthy subjects (4 F/7 M) were included by advertisements in university departments. Mean ± SD, (range) age 31.5 ± 16.2 years, (22-67), and weight 84.5 ± 12.7 kg, (75-110). Lacking previous data for power calculation, a cohort of 10-15 was deemed reasonable. The subjects were nonsmokers and abstained from alcohol and caffeine-containing beverages from the day prior to the experiment. To ensure normovolemia, the participants were encouraged to have a normal breakfast, including fluid (e.g., milk products and juice) ad libitum. No fluid was administered during the study. The volunteers were informed in writing and verbally about the study and signed a consent form.

| Equipment
The electrocardiogram (ECG) was sampled at 1000 Hz. Finapres (Finapres Medical Systems) continuously and noninvasively measured cardiovascular variables. Based on the Modelflow developed by Peñáz and Wesseling, brachial artery pressure is reconstructed from a reverse transfer function applied to finger arterial blood pressure measured by clamping finger arteries (Bos et al., 1996;Guelen et al., 2003;Peñáz et al., 1976;Wesseling et al., 1993). The equipment calculates SV, MAP, systolic and diastolic blood pressure (SBP, DBP, respectively) adjusted to the phlebostatic axis using the heart reference system (HRS).

| Procedure
The procedure strictly followed the recommendations of Monnet and Teboul (2015). The subject was placed K E Y W O R D S autonomic nervous system, cardiac output, heart rate variability, passive leg raising, preload responsiveness, stroke volume comfortably on an operating table in a semi-recumbent position with the legs horizontal (SR1). Baseline values of ECG, SV, CO, HR, MAP, SBP, and DBP were recorded beat-by-beat for 5 min. Next, the leg plate was elevated to 45° and the breastplate leveled (PLR). Recordings continued for 10 min before the position was returned to baseline semi-recumbent for a final 5-minute recording of said variables (SR2) (see Figure 1).

| Hemodynamic variables
Representative 5-minute sequences (250-300 samples) were selected from the Finometer recordings (HR, SBP/ DBP, MAP, SV, and CO) and ECG. Sequences were visually assessed as stable and free of artifacts.

| Heart rate variability
HRV is a description of the minute variations in the RR interval of the ECG. The spectral analysis dissects the vector of RR intervals as a composite of sinusoidal functions. The intervals are subjected to a Fourier transformation from which primarily the frequencies, phase angle, power, and amplitudes are obtained. In this pilot study, ECG data were exported in the European Data Format (EDF) to Kubios Premium 3.0.1 (Kubios Oy, www.kubios. com) (Figure 2), and the following spectral variables were extracted and reported from automatically corrected 5min samples of ECG during SR1, PLR, and SR2: high (HF, 0.15-0.4 Hz) and low (LF, 0.04-0.15 Hz) frequency power of the RR interval and the composite indices of parasympathetic (PNS) and sympathetic (SNS) activity (Tarvainen et al., 2021). PNS increases HRV and is considered a major contributor to the HF component. Conversely, the LF component of HRV is conventionally considered to include both SNS and PNS influence with SNS activity likely being the dominant (Iwase et al., 2017;Malik, 1996;Stick, 1996), accepting that HRV may be influenced by HR itself. The LF/HF ratio accentuates ANS-engendered changes.

| Statistics
All subjects provided analyzable data. After inspection and robust regression and outlier removal (ROUT; Motulsky & Brown, 2006), the normal distribution of the data was assessed using the Kolmogorov-Smirnov test. The differences between SR1 and PLR were tested using paired t-tests for HR, MAP, SV, and CO. Further analyses included one-way paired Gaussian analysis of variance (ANOVA) of mean values, as warranted by the normality of data (Sullivan et al., 2016). Statistical significance was defined as p < 0.05 (GraphPad Prism ver. 8.4 and 9.4.1). In view of the multiple significant results, post hoc power calculation was waived after consultation with in-hospital statistical expertize (Zhang et al., 2019).

| Heart rate variables
During PLR, LF power decreased, whereas HF increased; that is, the LF/HF ratio decreased ( Figure 4).The PNS index increased from SR1 to PLR (−0.141 to 0.996 a.u.; p < 0.0001), while the SNS index decreased (from 0.808 to −0.177 a.u.; p < 0.001; Figure 5). In addition, all HRV variables returned to baseline when subjects returned to SR2. The changes in the low and high frequencies in normalized units, as well as the LF/HF ratio, indicated a relative increase in parasympathetic and decrease (withdrawal) in sympathetic power during passive leg raising.

| Results
The relationship between PLR and ANS was studied in 11 normovolemic subjects who underwent the F I G U R E 1 Topological changes during PLR maneuver. PLR, passive leg raising; SR1, initial semi-recumbent position; SR2, final semi-recumbent position. standardized PLR test. Hemodynamic variables changed in response to PLR by increasing SV and decreasing MAP and HR, thereby maintaining CO. HRV variables reflecting SNS and PNS changed dominance from SR1 to PLR, and in reverse from PLR to SR2. Thus, an initial predominant SNS in response to draining of CBV was supplanted by a relative increase in PNS (withdrawal of SNS) during PLR. This ran in parallel with the increase in SV which may be seen as a response to the augmentation of CBV.

| Equipment and methodology
The PLR has been promulgated as a dependable and innocuous assessment of preload responsiveness in the ICU and recovery room. Its exact execution was honed in a study by Jabot showing that the sequence "semirecumbent to PLR" induces a larger increase in cardiac preload than "supine to PLR" and thus "SR to PLR" may be preferred for predicting fluid responsiveness (Jabot et al., 2009). This was later codified by Monnet and Teboul (2015). This study F I G U R E 2 Example of recording of RR intervals during SR1, PLR, and SR2. The two nadirs at approximately 11.24 and 11.35 mark the transition between positions F I G U R E 3 Mean and SD of individual measurements of MAP, HR, SV, and CO during the three phases. Only the changes in CO were insignificant. Ns: p > 0.05; **p ≤ 0.01; ***p ≤ 0.001 adhered to the Monnet & Teboul procedure to maintain comparability with the majority of studies on the subject of volume responsiveness.
Finapres was chosen as a representative of the group of minimally invasive monitors applying pulse contour analysis (PCA) incorporating the Peñáz/Wesseling Modelflow and physiocal algorithms (Geerts et al., 2011). The Finapres has been shown to agree satisfactorily with other evaluations of central hemodynamic variables (Broch et al., 2012;Rang et al., 2007;van der Spoel et al., 2012), specifically compared to thermodilution CO during baseline and PLR (Geerts et al., 2011). In a similar study, Elwan et al. (2018) used transthoracic electric bioimpedance which to the present author seems to have minor penetration in the intensive care community. Concerns have been raised after Doppler documented vasodilatation and increased flow induced by baroreflex during PLR (London et al., 1989).
The Peñáz/Wesseling PCA Modelflow, however, is not based on flow measurement, but integrates the area under the arterial pressure curve from the onset to the dicrotic notch and relates this area to cardiac output through an algorithmic autocalibration. Thus, the algorithm accounts for changes in arterial tone and compliance.
The ECG was recorded for HRV analysis in the spectral aspect. This is often applied when associations between ANS activity and clinical conditions are evaluated, for example, as a prognostic index for metabolic syndrome, heart failure, and neuroprognostication (Henden et al., 2014;Honoré et al., 2019;Vistisen et al., 2014). The reflection of the ANS subdivisions in the powers of LF, HF, and the LF/HF ratio is straightforward (with reservations noted above) and generally available in digital signal analysis applying fast Fourier transformation (FFT). Malik (1996) recommended a minimum sampling frequency of 250 to 500 Hz. Indisputably, the higher the sampling frequency, the higher the resolution and the greater the chance of discovering the miniscule changes in the RR interval. Simultaneously, it is of importance whether focus is on absolute or normalized units as suggested by Malik (1996) and Malik et al. (2019). Two early studies by McHugh bears witness to this: McHugh et al. (1994McHugh et al. ( , 1997. The first report dealt with a study of positional changes and spectral components in healthy volunteers and the second in postoperative CABG patients. Noteworthy is the use of absolute power of the elements of the spectral convolute and the very low sampling frequency of the electrocardiographic recording: 200 Hz. This may have contributed to the conclusion that positional changes did not induce changes in ANS responses. Furthermore, the ECG signal is prone to error and artifacts. Thus, the Kubios analysis program offers degrees of filtering. In this study, a built-in automatic, accurate algorithm for detecting artifacts (missed, extra, and misaligned beat detections) as well as ectopic beats was applied. This feature was not used in the studies by McHugh, nor the F I G U R E 4 LF, HF, and LF/HF in normalized units during SR1, PLR, and SR2. Differences between positions SR1/2 and PLR are significant. *p ≤ 0.05; **p ≤ 0.01 F I G U R E 5 Individual PNS and SNS indices during the three phases. A significant increase was seen in PNS during PLR concomitant with a decrease in SNS. Ns: p > 0.05; **p ≤ 0.01; ****p ≤ 0.0001 recent publication by Sejersen et al. (2022) probably explaining the lack of significant results. Notably, the road from registration of the electrical activity of the heart to the presentations of an ECG and final results of the FFT passes several stations in digital signal analysis in the shape of electrical components, analog-to-digital conversion, filtering, and analytical algorithms. If this sequence creates aberrant results, they may legitimately (from a statistical point of view) be removed by application of an outlier removal algorithm. ROUT was applied in this study and may be part of the successful demonstration of differences in contradistinction to studies referred to above. Gaffney et al. (1982) published a study noticing only a small, transient increase in stroke volume during PLR in healthy volunteers. The effect was absent after 30 to 45 min. In 1988, Wong demonstrated a 3% increase in CI in a cohort of patients. Results were reported for 5 min post-PLR. The study was followed in 1989 by a study including a fixed volume of 500 ml hemorrhaged blood in a cohort of healthy subjects, demonstrating a slight increase in cardiac and stroke index during PLR post-hemorrhage (Wong et al., 1989). Discussions in these three early studies of the effect of PLR centered on the gravitational dislocation of volume and the autonomic nervous reactions, although these were not actively monitored, to the maneuver. Anderson, following Wong's publication, noted that "The clinical significance of changes in cardiovascular function, after injury and hemorrhage, must ultimately lie in their influence on our ability to deliver oxygen to the tissues and our monitoring of this treatment" (Anderson, 1991). This is a tribute to Bernard's concept of the "milieu interieure" (Bernard, 1878) and Cannon's "homeostasis" (Cannon, 1939). According to Guyton, the cardiovascular system has "at least" three compensatory mechanisms (cp. homeostasis) offsetting the effects of changes in blood volume (here interpreted also as "changes in stressed volume or CBV"): nervous reflexes, stress relaxation/recovery and readjustment of blood volume by renal secretion/reabsorption and transcapillary autotransfusion/extravasation/lymphatic retransfusion via the thoracic duct (Guyton, 1963). The mechanisms have different time constants, thus reaching saturation after different time courses-reflexes acting fast, stress relaxation/recovery within minutes to hours and renal, lymphatic and transcapillary mechanisms within days. The acute events elicited by changing position from SR1 to PLR may be envisaged as an immediate increase in CBV manifesting as an increase in mean systemic filling pressure (MSFP). MSFP enters the term for venous return pressure, VRdP = MSFP-RAP. Depending on heart efficiency, the heart may maintain a constant RAP and the increased venous return increases CO. CO increases MAP which stimulates the aortic and carotid pressoreceptors, inhibits the SNS and activates the PNS, induces vasodilation, and decreases HR. Two studies demonstrate the importance of heart efficiency in the responses to PLR: Thomas and Shillingford (1965) presented measurements of arterial pressure, CO, HR, and SV in patients with presumed normal cardiac function and patients having suffered a myocardial infarction 2-6 weeks prior to the study. Measurements were made in 20° anti-Trendelenburg position and 3-6 min after 60° PLR. Subjects without heart disease increased SV by 35% ± 6.4%, whereas patients with a history of MI increased by 13% ± 19.9% (note: SD including instances of decreasing SV in response to PLR). Similarly, Si 53 years later related the PLR threshold values of ΔCO and ΔSV to Global Ejection Fraction (GEF is a transpulmonary thermodilution-derived index of the systolic function, [Litton & Morgan, 2012]) in two groups, GEF≥20% and GEF < 20%. The high-GEF group showed a 12% threshold of ΔSV versus 8% in the low-GEF group and ΔCO of 7 versus 6% (Si et al., 2018). In this study, 9/11 subjects increased SV as a reflection, reasonably explained by increased VRdP and normal heart efficiency and counteracted an increase in CO by lowering MAP and HR.

| The short-lived response to PLR
This study focused on differences in macrohemodynamics and HRV variables between positions of presumed equilibrated states, SR1, PLR, and SR2. This was borne out of the deliberation that FFT is not well suited for following rapid changes as the algorithm needs up to 5 min of registration to deliver reliable results. It is well-known that CO immediately after change of position increases and subsequently decreases. This has led to the recommendation that only fast reacting CO measurement methods should be employed (Monnet & Teboul, 2008, even finding its way into a renowned textbook by Marik (2015). One of the first studies was performed by Gaffney et al. (1982) who studied the time course of changes in SV/CO in healthy volunteers following PLR. Measurements were performed 20 s and 7 min after taking up the 60° PLR position. CO and SV increased significantly in the 20″-measurements but returned to baseline values within 7′. The speedy disappearance of induced changes is readily explained by reference to vascular stress relaxation as demonstrated by Prather and Guyton (1969). If a PLR-induced endogenous volume bolus has a duration of <7 min, Hilton and Bellomo (2012) raised the question for how long does an exogenous fluid bolus affect macrohemodynamics? It was answered by Farkas (2019) and Nunes et al. (2014): neither the endo-nor the exogenous fluid bolus have a lasting effect in fluid responders.
Another question concerns the role of ANS in producing the concordant, immediate changes in SV and CO. Frye and Braunwald (1960) published a study with the aim of determining whether the circulatory response to acutely induced hypervolemia were modified by reducing the activity of the ANS by means of ganglionic blockade. Seven subjects underwent serial phlebotomy to a cumulated volume of 1.5 L. Within 2 days after the last phlebotomy, the volume was returned in approximately 80 min. No intervention affecting ANS was made. Hemodynamics (HR, CO, and intra-arterial BP) were recorded before transfusion, immediately after completion and 15 min later. The phlebotomy sequence was repeated. Before transfusion of exsanguinated blood, an infusion of trimetaphan was instituted counteracting cholinergic transmission at the nicotinic receptors of the autonomic ganglia blocking the SNS and PNS. The infusion targeted a decrease in SBP of 40 mmHg. CO was determined by the indicator dilution technique. The remarkable differences were related to the absence of autonomic reflexes during ganglionic blockade, summarized in the conclusion: "Experiments in which hypervolemia is induced acutely in intact human subjects may not be ideally suited for determining the applicability of Starling's law of the heart to man. The presence of an intact autonomic nervous system results in failure of a significant fraction of the infused fluids to augment the volume of blood within the heart and lungs, and the stimulation of myocardial contraction provided by an increased end-diastolic fiber length cannot result." In this study, a discordance between SV and CO increases was noted: SV pointed to intact volume responsiveness, whereas CO did not. Possibly the ANS activity implied a counterregulation maintaining DO 2 in a situation where VO 2 was constant as no physical activity was present and by implication constant CO. In the Frye study, both SV and CO remained stable for the considerably longer period of transfusion, 80 minutes, compared to the 10 min of PLR in the present setting.
Looking further, studies identifying preload responsiveness based on PLR vary according to the chosen variable. Some favor SV (Biais et al., 2009;Preau et al., 2010;Thiel et al., 2009) while others use CO (Cherpanath et al., 2016;Monge Garcia et al., 2012;Monnet et al., 2012), both with a cut-off of 13%-15% based on receiver operating characteristics (ROC) analysis. Two meta-analyses demonstrated almost equal numbers of studies using CO (as aortic blood flow) and SV for the assessment of preload responsiveness. Studies using CO as the arbiter of preload responsiveness have likely been carried out in patients with attenuated autonomic and homeostatic reflexes, whereas in healthy volunteers, the differential responses of CO and SV are revealed as CO is preserved in face of unaltered "milieu intérieure." Thus, from a clinical perspective, it is of interest to gauge the interaction between central hemodynamic variables and autonomic control during PLR in patients with septic and acute heart failure, as well as in patients exposed to sedation or anesthesia, that is, sympatholytic states. If SV and CO increases in response to PLR are associated with attenuation of the SNS index, the responses may be induced by vasoplegia, increased pooling in an unresponsive distal vascular bed during SR1, and unabated augmentation of CBV increasing MSFP in a relatively noncompliant central venous circulation during PLR. This "preload responsiveness" should be interpreted cautiously when administering external fluid and assessing the need for volume expansion rather than correction of vasodilatation.

| Limitations
The number of volunteers was limited to 11, which was sufficient for probing the PLR and ANS. The significant results invite a larger study including both healthy persons and patients. This study addressed the relationship between HRV and PLR in healthy volunteers and directed attention to the discordant responses in SV and CO. To become clinically relevant, patients should be incorporated and the ANS should be pharmacologically manipulated. It is a limitation that a supine position was not included in the protocol for exploration of the mechanisms involved in the PLR procedure, but the study specifically addressed the recommendations provided by Monnet and Teboul (2015). From a pragmatic and clinical feasibility point of view, it is outside the scope to look for changes in CBV, for example, by regional electrical admittance (Cai et al., 2000), echocardiography (Brothers et al., 2014), and transpulmonary thermodilution for the determination of CO, intrathoracic blood volume, and global end-diastolic volume (Reuter et al., 2010).

| CONCLUSION
In conclusion, the gravitational influence on the CBV inherent to the PLR maneuver elicits a modulatory autonomic signal effecting central hemodynamic responses, preserving homeostasis of CO. In terms of SV, normal subjects appear volume responsive, whereas stable CO results from the homeostatic mechanisms of the human body.

AUTHOR CONTRIBUTIONS
Søren Søndergaard designed the study, performed the analyses, and wrote the first and final drafts of the manuscript.